Transcript Indirect (negative) staining

Indirect (negative) staining
• Most biological materials show
little contrast with their
surroundings unless they are
stained.
• In the case of light microscopy,
contrast can be enhanced by using
colored stains which selectively
absorb certain wavelengths.
Indirect (negative) staining
• To heighten the contrast between bacteria
and the background, use is made of
electron-dense "stains".
• These are usually compounds of heavy
metals of high atomic number, that serve to
scatter the electrons from regions covered
with the stain.
• If bacterial particles are coated with stain
(positive staining), fine detail may be
obscured.
Indirect (negative) staining
• Negative staining overcomes this
problem by staining the background
and leaving the bacteria relatively
untouched.
• The negative stain is molded round the
bacterial particle, outlining its
structure, and is also able to penetrate
between small surface projections and
to delineate them.
Indirect (negative) staining
• The acidic dye nigrosine will be used to
visualize the capsule or sheath that
surrounds some bacteria in a process called
negative staining.
• Capsules are composed primarily of
polysaccharides or glycoproteins and are
gelatinous in texture.
Indirect (negative) staining
• They are readily destroyed by heating and
hence direct staining methods cannot be
utilized.
• In general, the size and shape of
microorganisms is often less distorted with
indirect staining procedures, especially
when sampled from a broth culture
Indirect (negative) staining
• Therefore negative staining is useful
whenever the morphology of individual
bacteria is in question.
• Morphology can often be determined with
confidence with only the high dry lens.
Consider that this procedure does not
necessarily kill the organism, so be careful.
Indirect (negative) staining
• After preparing a clean, greaseless slide, a
small drop of nigrosine is mixed with a
small drop from a broth culture or with a
quantity of dry material.
• The drop is spread across the slide using the
edge of another slide as a spreader. This
same procedure is used for blood smears.